The central theme of this project is a study of lipid-protein interactions in biological membranes with the eventual aim of understanding the molecular details of lipid-protein associations. The strategy is to introduce spin label analogues of specific, naturally occurring membrane phospholipids into preparations containing a single kind of functional protein, and to study lipid mobility and distribution by electron spin resonance. Initial work utilizing single tail lipids differing in charge on the polar group show that the eel Na,K-ATPase segregates negatively charged amphiphiles with a four-fold greater affinity than the positively charged analogous molecules. This specificity involves an unknown, but probably small, number of binding sites. Currently, our objectives are characterization of the specific phospholipid binding sites of several transmembranous proteins. Specifically, our objectives are to study the integral membrane proteins Na,K-ATPase and cytochrome oxidase reconstituted in defined lipids. By correlating ESR data, NMR data, activity measurements, electron microscopy and lipid analyses, it will be possible to characterize the specific interactions of these membrane proteins with phospholipids differing in head group.